INTRODUCTION:

Papain, a proteolytic enzyme found naturally in papaya (Carica papaya L.) fruits. It’s able to break down organic molecules made of amino acids, known as polypeptides, therefore thus plays a crucial role in drug designs, industrial and pharmaceutical preparations [1–3].

Infections caused by multidrug-resistant (MDR) pathogens have become challenging over the past years, particularly in the developing countries, and are associated with high rates of morbidity and mortality [4–6]. In 2017, the World Health Organization (WHO) declared that Enterobacteriaceae, especially extended-spectrum β-lactamase (ESBL) producing K. pneumoniae, which listed as priority pathogens that urgently required new antibiotic development due to their widespread resistance [7,8]. Fluoroquinolones are currently among the most heavily prescribed antimicrobials in the world because of their spectrum of activity, their pharmacokinetic profiles, and their generally good tolerance [9,10]. Those antibiotics target DNA gyrase and topoisomerase IV with varying efficiency in different bacteria and inhibit their supercoiling control within the cell, resulting in impaired DNA replication and cell death [11,12].

K. pneumoniae, in the last two decades, has become a relevant healthcare-associated pathogen, and the causative agent of approximately 14–20% of the infections related to the respiratory tract, lower biliary duct, surgical wounds and urinary tract [13]. The resistance of Klebsiella pneumoniae to the beta-lactam antibiotics is due to the bacterium production of special enzymes such as carbapenemases, and New Delhi Metallo-beta-lactamase [14,15]. Different mechanisms are responsible for resistance to antibiotics in bacteria such as Inactivation of antibiotics, Target site modiﬁcation, Prevent drug uptake, Efﬂux pumps (EP) and Bioﬁlm formation [16–19]. As increased acquired resistance to conventional antibiotics is observed, it is rational to attempt combination therapy of standard antibiotics with plant extracts that possess bio-enhancing activity to attain bactericidal synergism [20], Use of such combination therapy against resistant bacteria may lead to new options for the treatment of infectious diseases [21]. Combination therapy can be used for the expansion of the antimicrobial spectrum, prevention of the emergence of resistant mutants, minimizing the toxicity [16].

Rotavirus is a member of the Reoviridae family, at which since their discovery in 1973, rotaviruses have been identified as the most common cause of dehydrating diarrhea in children [22–24]. In 2009, the WHO recommended the rotavirus vaccine for infants, especially in countries where diarrheal deaths account for ≥ 10% of mortality among children aged < 5 years, at which the introduction of the vaccine is strongly recommended [25]. Two live, attenuated, oral rotavirus vaccines − Rotarix (monovalent rotavirus vaccine [RV1], GSK Biologicals, Rixensart, Belgium) and RotaTeq (pentavalent rotavirus vaccine [RV5], Merck & Co. Inc., Kenilworth, NJ) − are WHO-prequalified and currently licensed in > 100 countries. But due to the high cost of these vaccines and logistic issues, thus the development of cheap and effective antiviral agents is indispensable to combat this virus [22,25].

According to the previous studies, the antibiofilm activity of proteases enzymes such as bromelain and papain enzymes was demonstrated against strains of Klebsiella pneumoniae, but no antibacterial activity was detected for both enzymes [5]. On the other hand, certain concentrations of papain enzyme showed a good activity to inhibit and eradicate Klebsiella pneumoniae biofilms [26]. Therefore, this study was designed to test the cytotoxicity of effective concentrations of papain enzyme, combining with antibiotics, and test its antiviral activity.

MATERIALS AND METHODS:

Microbial strains and antibiotics used:

Ten biofilm-producing multi-drug resistant (MDR) K. pneumoniae strains were recovered [5,21,26]. Strains were previously identified by conventional microbiological methods and confirmed by MALDI-TOF/MS using Bruker Biotyper 3.1 software. Ciprofloxacin and Levofloxacin antibiotics were used. Antibiotic susceptibility testing was done previously.

Enzyme preparation:

Papain from Carica papaya (≥3 U/mg) was obtained from Sigma-Aldrich (St. Louis, USA). Dilution of 100 mg/ml was prepared according to the manufacturer's instructions and then sterilized by filtration before assays.

Cytotoxicity evaluation:

Evaluation of cell morphology was done by using inverted light microscopy [27] with modifications, MA104 cell lines (2x10⁵ cells/ml) were seeded in 96-well tissue culture plates (Corning, US). After incubating the seeded plates for 24 h at 37ºC in a humidified 5% CO₂ atmosphere, the culture medium was discarded from each well and replaced with 200µl of compound dilutions per well prepared in culture medium. For cell controls, 200µl of culture medium without compounds was added. All culture plates were incubated in a humidified 5% CO₂ atmosphere at 37ºC for 72 h. Cell morphology was checked daily for microscopically detectable morphological changes, such as cell rounding and shrinking, loss of confluency, and cytoplasm granulation and vacuolization. Morphological alterations were scored and the 100% safe dilutions against the cell morphology were selected for the antiviral assay.

Effect of papain concentration on antibiotic activity by micro-broth dilution method:

Minimum inhibitory concentration (MIC) of antibiotics alone and in combination with a fixed antibiofilm concentration of papain enzyme (3mg/ml) was done using the broth micro-dilution method [5]. In each well of 96 microtiter plate, 100µl of bacterial suspension were added to 100µl of fixed enzyme dilution at which the final dilutions of ciprofloxacin and levofloxacin concentrations were 0.25- 64µg/ml and 0.25-16µg/ml respectively. Visually the MIC was determined which was the well with no visual bacterial growth.

Antiviral activities of tested compound by measurement of cytopathic effect:

For tissue culture infection dose (TCID₅₀) determination, the safe dilution of the tested compound was used to be evaluated against RV infection. 10-fold dilutions of activated RV SA-11 were prepared in cell culture medium then 100 μl of each viral dilutions 10^-4-10^-9 were incubated with 100 μl of the tested compound for one hour at 37^oC in CO₂ incubator. Virus dilutions either with or without compound were added into four parallel wells. All plates were incubated at 37^oC in CO₂ incubator for 72 h, then the cytopathic effect was observed under an inverted microscope and virus titration was calculated and expressed as 50% TCID₅₀ by using Spearman Kärber method [28]. The reduction in virus titer was calculated as differences between the values of the treated and untreated virus.

RESULTS AND DISCUSSION:

Antimicrobial resistance (AMR) is of significant concern in developing nations due to over-use of antimicrobial agents, widespread availability of counterfeit or substandard drugs, and poor infection control measures [29,30]. Treating bacterial infections will likely become more challenging in the future. We need to protect the antibiotics we already have, develop new ones, and redouble our efforts to generate novel therapies against bacterial pathogens [31]. Recently, we concluded that proteases enzymes such as bromelain and papain had antibiofilm activity against resistant strains of clinical Klebsiella pneumonia, but despite this anti-virulence activity, no antibacterial effect was detected neither for bromelain nor papain enzyme [5,26].

In our study, high toxicity on MA104 was observed using all papain concentrations used, except for the lowest concentration which was the least toxicity (Data not showed). These results are in line with another study where papain exhibited negative results for all the performed tests (Cytotoxicity assay, Growth inhibition test, WP2-Mutoxitest, and Plasmid-DNA treatment, and agarose gel electrophoresis) [32]. On the other hand, it was reported that 1000μg/ml of papain has a maximum (85%) cytotoxicity effect against liver cancer cell line hepG2 [33]. Also, MTT assays showed that papain at concentrations of up to 100μg/mL did suppress cell growth only to slightly below untreated control [34]. In addition, another proteolytic enzyme such as bromelain was afforded substantial anti-cancer potential which suggested as a potential prospect for anti-cancer agent besides only an additive in chemotherapy [35].

In this study, the combination effect of papain enzyme (3 mg/ml) with quinolone antibiotics represented in Table 1, showed that the antagonistic effect resulted when combined with ciprofloxacin (3 strains). But for levofloxacin, antagonistic effect showed in one strain. On the other hand, all the other strains showed no effect before and after the combination assay.

Table 1: Effect of papain combination with antibiotics against Klebsiella pneumoniae strains

Strains	MIC of Antibiotics (µg/ml)
	Ciprofloxacin		Levofloxacin
	Without enzyme	With enzyme	Without enzyme	With enzyme
Kp1	*	32**	8	8
Kp2	*	˃ 64	˃ 16	˃ 16
Kp3	*	32**	˃ 16	˃ 16
Kp4	*	16	˃ 16	˃ 16
Kp5	*	32	4	8**
Kp6	*	32**	˃ 16	˃ 16
Kp7	*	64	8	8
Kp8	*	16	4	4
Kp9	*	16	˃ 16	˃ 16
Kp10	*	˃ 64	8	8

*MIC results of ciprofloxacin were previously published [5], where the MIC repeated in this assay under the same conditions and the same results observed.**Antagonistic activity.

In the last few years, many studies tried to study the antimicrobial activity of various enzymes against a wide range of pathogenic and non-pathogenic microorganisms. Glucose oxidase from A. niger showed anti-bacterial activity against P. multicida and S. aureus [36]. Anjos et al. (2018) reported that papain and bromelain exerts an antimicrobial potential against strains of Alicyclobacillus [37]. The purified chitinase produced by A. terreus was observed to inhibit the growth of Aspergillus niger, Aspergillus oryzae, Penicillum oxysporium, Rhizocotonia solani, Candida albicans and Fusarium solani, while did not inhibit the growth of Rhizopus oryzae [38].

In order to expand the antimicrobial spectrum, reduce toxicity and decrease the antimicrobial resistance, antibiotic agents combinations were applied [39]. Among Lysozyme and serratio-peptidase enzymes, Penta et al. (2010) observed that serratio-peptidase was found to inhibit the growth of both S. aureus and K. pneumonia during combination with gatifloxacin, rifampicin, streptomycin and ampicillin showing synergistic effect [40]. Trypsin and chymotrypsin were found to be effectively inhibiting the growth of pathogenic bacteria related to cow mastitis by destroying bacterial external structures [41].

Finally, concerning the antiviral assay, although, Carica papaya extracts were reported for their anti-HIV-1 activity at which, methanol and aqueous extracts showed activity as anti-HIV-1 agents [42], but about papain effect on rotavirus in our study, no antiviral activity was shown against rotavirus infection in vitro when various concentrations (10^-4-10^-9) of virus prepared from viral stock of 10⁶ TCID₅₀.

CONCLUSION:

According to our results, although the papain enzyme had strong antibiofilm activity against Klebsiella strains, no antibacterial or antibiotic enhancing activity was detected. The tested compound has high toxicity on MA104 and thereby no antiviral activity was shown against rotavirus infection in vitro. Therefore, through our study and based on the results of the toxicity test, we suggest that the tested concentrations of the enzyme should not be used for pharmaceutical purposes, but based on its ability to inhibit and eradicate biofilms of Klebsiella, we can use it for surfaces sterilization purposes, but this also needs further studies.

AUTHORS CONTRIBUTIONS:

This work was carried out in collaboration among all authors. Mabrouk MI and Mohamed SH; Designed the research. Mohamed SH and Mohamed WS; Performed bacterial in vitro experiments and wrote the paper. Shaheen MNF and Elmahdy M,; Designed and performed cytotoxicity and antiviral experiments and wrote the paper. Mabrouk MI; Supervised the research. All authors read and approved the final manuscript.

COMPETING INTERESTS:

Authors have declared that no competing interests exist.

FUNDING: None

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Received on 23.10.2019 Modified on 31.12.2019

Asian J. Res. Pharm. Sci. 2020; 10(1):06-10.

DOI: 10.5958/2231-5659.2020.00002.8